Methods for measuring transporter activity using budding baculoviruses that do not express endogenous transporters on their envelope have a low background level and can measure the target activity with a high sensitivity. Such methods can be used to measure functional changes due to transporter SNPs over a more extensive range of substrates, and can be applied to tailor-made therapies.
|
11. A purified baculovirus, the envelope of which comprises a mammalian transporter having transporter activity.
14. A method for measuring the activity of a mammalian transporter, the method comprising
(a) providing a budding baculovirus the envelope of which comprises a mammalian transporter having transporter activity; and
(b) measuring the activity of the mammalian transporter.
17. A method of screening for a substance that inhibits or promotes transport activity of a mammalian transporter, the method comprising
(a) providing a budding baculovirus the envelope of which comprises a mammalian transporter having transporter activity;
(b) contacting the transporter with a test substance, and
(c) selecting a substance that inhibits or promotes the transport activity.
1. A method for preparing a budding virus expressing a mammalian transporter having transporter activity, the method comprising
(a) culturing a host cell that (i) is infected with a budding virus that comprises a gene encoding the transporter and (ii) is recombinantly expressing the transporter;
(b) expressing the transporter on the envelope of a budding virus released from the host cell; and
(c) harvesting the released virus, wherein the transporter on the envelope of the released virus has transporter activity.
3. The method of
4. The method of
7. The method of
8. The method of
9. The method of
10. The method of
12. The baculovirus of
15. The method of
18. The method of
21. The method of
22. The method of
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The present invention relates to methods for expressing transporters having transporter activity, where the methods comprise using transporter-encoding genes to express transporters on the envelope of budding viruses. In addition, the present invention relates to viruses that express transporters having transporter activity, methods that use these viruses for measuring the transporter activity, and methods of screening for substances that inhibit or promote the transporter activity of the transporters.
Mammals must take in nutrients from outside the body, and many transporter proteins (transporters) are known to exist in mammalian cells. These transporters mainly act to transport substances essential to the maintenance of life (amino acids, sugars, and such) into cells. In the physiological environment, cells often have multiple transporters that transport the same substrate. In these cases, the individual contribution of transporters to cellular uptake can be estimated using kinetic analysis (calculation of Km, Vmax, and so on; e.g., Wright E. M., Am. J. Physiol. Renal Physiol., 2001, 280: F10-18). Thus, identification of transport substrates and kinetic analysis of transporters are extremely important for revealing their physiological function and their potential in drug delivery.
Currently, methods for analysing transporter function use the following resources as materials: (1) primary cultured cells and cell membrane vesicles (such as lung cells and brush border membrane vesicles) comprising transporters isolated from living bodies; (2) cell lines derived from transporter-comprising cancer cells and so on (such as Caco-2 cells); (3) mammalian cells introduced with transporter genes (such as LLC-PK1 cells and MDCK cells) and Xenopus oocytes; and (4) insect cell membranes (such as Sf9 cell membranes) in which transporters have been expressed using baculovirus expression systems. Of these, mostly used are gene expression systems from mammalian cells and Xenopus oocyte cells. However, even in mammalian and Xenopus oocyte cells introduced with transporter genes, activities from endogenous transporters can be detected, thus elevating background levels (Kanai Y. et al., J. Clin. Invest. 93: 397-404 (1994); Kekuda R. et al., J. Biol. Chem. 271: 18657-18661 (1996); Kekuda R. et al., Am. J. Physiol. 272: G1463-1472 (1997); Yabuuchi H. et al., J. Pharmacol. Exp. Ther. 286: 1391-1396 (1998); Hatanaka T. et al., J. Clin. Invest. 107: 1035-1043 (2001)). For this reason, in some types of transporters, there are reports that describe an activity ratio of only two between cells introduced with genes and those not introduced with genes (parent cell lines). Carrying out kinetic analysis can be problematic in such gene-introduced cells with a low activity ratio.
In Xenopus oocyte cells introduced with transporter genes, transporter activity can be measured using electrophysiological methods. In transporters driven by Na and H ions, and substrates having an electric charge at physiological pH, transporter activity can be detected by measuring the electrical current caused by substrate transport. However, measuring transport activity is difficult when there is no driving force and also when substrates are electrically neutral at physiological pH. Kinetic analysis is also difficult in cases where transporter activity is observed but only a weak current can be detected. In addition, since electrophysiological methods require specific equipment, they are not simple or convenient.
The activity and substrate specificity of transporters that transfer drugs into cells has been reported to influence the drug's bioavailability (for example, Ganaphthy, Leibach, Curr. Biol 3: 695-701 (1991); Nakashima et al., Biochem. Pharm. 33: 3345-3352 (1984); Friedman, Amidon, Pharm. Res. 6:1043-1047 (1989); Okano et al., J. Biol. Chem. 261: 14130-14134 (1986); Muranushi et al., Pharm. Res. 6: 308-312 (1989); Friedman, Amidon, J. Control. Res. 13: 141-146 (1990)). In recent years, research on factors that fluctuate in vivo pharmacokinetics has clarified that drug-metabolising enzymes as well as drug-transporters influence the function of drugs in the body. Known drug-transporters include p-glycoprotein (Annu. Rev. Biochem. 58: 137 (1989)), multidrug resistance protein (Science 258: 1650 (1992); Cancer Res. 55: 102 (1995)), lung resistance protein (Ann. Oncl. 7: 625 (1996); Int. J. Cancer 73: 1021 (1997)), and organic cation transporter (Proc. Natl. Acad. Sci. USA 91: 133 (1994); Molec. Pharmacol. 51: 913 (1997)). Analysis of SNPs is being carried out for these drug-transporters in the same way as for drug-metabolizing enzymes. Transporter SNPs that bring about functional changes have been recently found. These SNPs are receiving attention as one of the factors causing fluctuations between individuals (Ryu S. et al., J. Biol. Chem. 275: 39617-39624 (2000); Tirona R. G. et al., J. Biol. Chem. 276: 35669-35675 (2001)). Currently, functional analysis of transporter SNPs mainly uses mammalian cells introduced with genes. However, this is speculated to be problematic for accurately detecting functional changes caused by SNPs in substrates having a low activity ratio compared to parent cell lines.
The present invention was made considering the above circumstances. An objective of the present invention is to provide methods for measuring the target transporter activity, which have a low background level and a high degree of sensitivity. In addition, another objective of the present invention is to provide methods of screening for substances that inhibit or promote the transport activity of transporters, using the above methods.
Since viruses have no fundamental need to self-reproduce, the present inventors speculated that there was no physiological value in taking up substances essential to maintenance of life. Thus, they focused on the assumption that endogenous transporters may not be expressed (or may be expressed in extremely low amounts) on viral envelopes. The method for measuring transporter activity using budding baculoviruses that do not express endogenous transporters on their envelopes are thought to have a low background level, and to enable a highly sensitive measurement of target activity. Furthermore, by using such methods, functional changes due to transporter SNPs can be measured for a broader range of substrates, and may be applied to tailor-made therapies.
Specifically, the present invention provides:
[1] a method for expressing a transporter having transporter activity, wherein the method comprises culturing a host infected with a recombinant virus that comprises a gene encoding the transporter, and expressing the transporter on the envelope of a budding virus released from the host;
[2] the method of [1], wherein the virus is a baculovirus;
[3] the method of [1] or [2], wherein the transporter is of a non-viral origin;
[4] the method of any of [1] to [3], wherein the transporter is a peptide transporter or an organic anion transporter;
[5] the method of [4] wherein the transporter is PepT1, PepT2, or OATP-C;
[6] a virus that expresses a transporter having transporter activity;
[7] the virus of [6], wherein the transporter is of a non-viral origin;
[8] the virus of [7] wherein the virus is a baculovirus;
[9] the virus of any of [6] to [8] wherein the virus is a budding virus;
[10] the virus of any of [6] to [9] wherein the transporter is a peptide transporter or an organic anion transporter;
[11] the virus of [10] wherein the transporter is PepT1, PepT2, or OATP-C;
[12] a method for measuring the activity of a transporter, wherein the method comprises expressing the transporter on a viral envelope;
[13] the method of [12] wherein the virus is a budding baculovirus;
[14] the method of [12] or [13] wherein the transporter is a peptide transporter or an organic anion transporter;
[15] the method of [14] wherein the transporter is PepT1, PepT2, or OATP-C;
[16] a method of screening for a substance that inhibits or promotes transport activity of a transporter, wherein the method comprises the following steps:
[17] the method of [16] wherein the virus is a baculovirus;
[18] the method of [16] or [17] wherein the virus is a budding virus;
[19] the method of any of [16] to [18], wherein the transporter is of a non-viral origin;
[20] the method of any of [16] to [19], wherein the transporter is a peptide transporter or an organic anion transporter;
[21] the method of [20] wherein the transporter is PepT1, PepT2, or OATP-C;
[22] the method of any of [16] to [21], which comprises immobilizing the virus on a support;
[23] the method of [22] wherein the virus is immobilized on the support through an antibody against an envelope protein expressed on the viral envelope; and,
[24] the method of [22] wherein the virus is immobilized on the support through a biotin-streptavidin reaction by biotinylating a protein expressed on the viral envelope.
The present invention relates to methods for expressing transporters having transporter activity, which methods comprise culturing a host infected with a recombinant virus that comprises a gene coding for a transporter, and expressing the transporter on the envelope of a budding virus released from the host. Herein, examples of a “transporter” include peptide transporters, amino acid transporters, and sugar transporters. More specifically, transporters such as those listed in Table 1 can be given as examples.
TABLE 1
Driving force/
Amino
Trans-
Transporter
transport type
acids
membrane
ncbi
Reference
4F2hc
LAT regulatory
529
1
P08195
Proc. Natl. Acad. Sci. U.S.A. 84
factor
(18), 6526-6530 (1987)
AE4
Cl/HCO exchange
945
14
AAK16733
Commun. 282 (5), 1103-1109
transport
(2001)
ATB0/ASCT2
Na/neutral amino
541
10
Q15758
J. Biol. Chem. 271 (31), 18657-18661
acid cotransport
(1996)
ATB0+
Na/neutral and
642
12
AAD49223
J. Biol. Chem. 274 (34), 23740-23745
basic amino acids
(1999)
cotransport
BAT1/b0+
Facilitated diffusion
487
12
P82251
Nat. Genet. 23 (1), 52-57 (1999)
AT
(amino acid)
BCRP
ATP/primary
655
6
AAC97367
Proc. Natl. Acad. Sci. U.S.A. 95
acive transport
(26), 15665-15670 (1998)
BSEP
ATP/primary
1321
12
AAC77455
Nat. Genet. 20 (3), 233-238
active transport
(1998)
BTR1
Cl/HCO exchange
891
14
AAK16734
Commun. 282 (5), 1103-1109
transport
(2001)
CNT1
Na/nucleoside
649
13
NP_004204
Am. J. Physiol. 272 (2), C707-C714
cotransport
(1997)
CNT2
Na/nucleoside
658
14
O43868
Am. J. Physiol. 273 (6 Pt 2),
cotransport
F1058-F1065 (1997)
CNT3
Na/nucleoside
691
13
NP_071410
J. Biol. Chem. 276 (4), 2914-2927
cotransport
(2001)
DRA/CLD
Cl/HCO exchange
764
P40879
Proc. Natl. Acad. Sci. U.S.A. 90
transport
(9), 4166-4170 (1993)
EAAC1
Na/acidic amino
525
12
NP_004161
Genomics 20 (2), 335-336
acid cotransport
(1994)
ENT1
Facilitated diffusion
456
14
NP_004946
Nat. Med. 3 (1), 89-93 (1997)
(nucleoside)
ENT2
Facilitated diffusion
456
14
AAC39526
Biochem. J. 328 (Pt 3), 739-743
(nucleoside)
(1997)
FORT
Folic acid
591
12
P41440
Commun. 206 (2), 681-687
(1995)
GAT1
Na/GABA
599
12
NP_003033
FEBS Lett. 269 (1), 181-184
cotransport
(1990)
GAT3
Na/GABA
632
12
P48066
Recept. Channels 2 (3), 207-213
cotransport
(1994)
GLUT1
Facilitated diffusion
492
12
NP_006507
Science 229 (4717), 941-945
(glucose)
(1985)
GLUT2
Facilitated diffusion
524
12
NP_000331
Proc. Natl. Acad. Sci. U.S.A. 85
(glucose)
(15), 5434-5438 (1988)
GLUT3
Facilitated diffusion
496
12
NP_008862
J. Biol. Chem. 263, 15245-15248
(glucose)
(1988)
GLUT4
Facilitated diffusion
509
12
NP_001033
J. Biol. Chem. 264 (14), 7776-7779
(glucose)
(1989)
GLVR1/PiT-1
Na/Pi
679
10
NP_005406
Cell Growth Differ. 1 (3), 119-127
cotransport
(1990)
GLVR2/PiT-2
Na/Pi
652
10
NP_006740
J. Virol. 65 (11), 6316-6319
cotransport
(1991)
LAT1
Facilitated diffusion
507
12
JG0165
Commun. 255 (2), 283-288
(amino acid)
(1999)
LRP
ATP/primary
896
NP_059447
Nat. Med. 1 (6), 578-582 (1995)
active transport
MCT1
H/organic anion
500
12
NP_003042
Genomics 23 (2), 500-503
cotransport
(1994)
MCT2
H/organic anion
478
12
O60669
J. Biol. Chem. 273 (44), 28959-28965
cotransport
(1998)
MCT3
H/organic anion
465
12
O15427
Biochem. J. 329 (Pt 2), 321-328
cotransport
(1998)
MCT4
H/organic anion
487
12
O15374
Biochem. J. 329 (Pt 2), 321-328
cotransport
(1998)
MCT5
H/organic anion
505
12
O15375
Biochem. J. 329 (Pt 2), 321-328
cotransport
(1998)
MCT6
H/organic anion
523
12
O15403
Biochem. J. 329 (Pt 2), 321-328
cotransport
(1998)
MDR1
ATP/primary
1279
12
AAB69423
Cell 47 (3), 381-389 (1986)
active transport
MDR3
ATP/primary
1279
12
P21439
EMBO J. 6 (11), 3325-3331
active transport
(1987)
MRP1
ATP/primary
1531
17
P33527
Science 258 (5088), 1650-1654
active transport
(1992)
MRP2
ATP/primary
1545
17
Q92887
Cancer Res. 56 (18), 4124-4129
active transport
(1996)
MRP3
ATP/primary
1527
17
NP_003777
Cancer Res. 57 (16), 3537-3547
active transport
(1997)
MRP4
ATP/primary
1325
12
NP_005836
Cancer Res. 57 (16), 3537-3547
active transport
(1997)
MRP5
ATP/primary
1437
12
O15440
Cancer Res. 57 (16), 3537-3547
active transport
(1997)
MRP6
ATP/primary
1503
17
O95255
Cancer Res. 59 (1), 175-182
active transport
(1999)
MRP7
ATP/primary
1492
17
Cancer Lett. 162 (2), 181-191
active transport
(2001)
NaPi-3B
Na/Pi
690
8
NP_006415
Commun. 258 (3), 578-582
cotransport
(1999)
NaSi-1
Na/Si
595
13
NP_071889
Genomics 70 (3), 354-363
cotransport
(2000)
NHE1
Na/H exchange
815
12
P19634
Cell 56 (2), 271-280 (1989)
transport
NHE2
Na/H exchange
812
12
NP_003039
Am. J. Physiol. 40 (2), 383-390
transport
(1999)
NHE3
Na/H exchange
834
12
NP_004165
Am. J. Physiol. 269 (1 Pt 1),
transport
C198-C206 (1995)
NPT1
Na/Pi
467
6-8
Q14916
Genomics 18 (2), 355-359
cotransport
(1993)
NPT2/NaPi-3
Na/Pi
639
8
NP_003043
Proc. Natl. Acad. Sci. U.S.A. 90,
cotransport
5979-5983 (1993)
Nramp2/DCT1
Na/Fe
568
12
P49281
Mol. Immunol. 34 (12-13), 839-842
cotransport
(1997)
NTCP2/ASBT
Na/bile acid
348
7
NP000443
J. Biol. Chem. 270 (45), 27228-27234
cotransport
(1995)
OAT1
Facilitated diffusion
550
12
NP_004781
Commun. 255 (2), 508-514
(organic anion)
(1999)
OAT2
Facilitated diffusion
548
12
NP_006663
(organic anion)
OAT3
Facilitated diffusion
568
12
NP_004781
Commun. 255 (2), 508-514
(organic anion)
(1999)
OAT4
Facilitated diffusion
550
12
AAK68155
J. Biol. Chem. 275 (6), 4507-4512
(organic anion)
(2000)
OATP-A
Facilitated diffusion
670
12
NP_066580
Gastroenterology 109 (4), 1274-1282
(organic anion)
(1995)
OATP-B
Facilitated diffusion
709
12
NP_009187
Commun. 273 (1), 251-260
(organic anion)
(2000)
OATP-C
Facilitated diffusion
691
12
BAA78639
Commun. 273 (1), 251-260
(organic anion)
(2000)
OATP-D
Facilitated diffusion
710
12
BAA89287
Commun. 273 (1), 251-260
(organic anion)
(2000)
OATP-E
Facilitated diffusion
722
12
BAA89288
Commun. 273 (1), 251-260
(organic anion)
(2000)
OCT1
Facilitated diffusion
554
12
NP_003048
Mol. Pharmacol. 51 (6), 913-921
(organic cation)
(1997)
OCT2
Facilitated diffusion
555
12
NP_003049
DNA Cell Biol. 16 (7), 871-881
(organic cation)
(1997)
OCT3
Facilitated diffusion
551
12
NP_035525
Genomics 55 (2), 209-218
(organic cation)
(1999)
OCTN1
H/organic
551
11
NP_003050
FEBS Lett. 419 (1), 107-111
cation
(1997)
OCTN2
Na/organic cation
557
12
O76082
Commun. 246 (3), 589-595
cotransport
(1998)
PGT
Facilitated diffusion
643
12
NP_005612
Commun. 221 (2), 454-458
(organic anion)
(1996)
rBAT
BAT1 regulatory
685
1
AAA81778
J. Biol. Chem. 268 (20), 14842-14849
factor
(1993)
SDCT1/NaDC-1
Na/dicarboxylic
592
8
NP_003975
Am. J. Physiol. 270 (4 Pt 2),
acid cotransport
F642-F648 (1996)
SGLT1
Na/glucose
664
14
NP00334
Proc. Natl. Acad. Sci. U.S.A. 86
cotransport
(15), 5748-5752 (1989)
SGLT2
Na/glucose
672
14
NP_003032
Am. J. Physiol. 263 (3 Pt 2),
cotransport
F459-F465 (1992)
SGLT3/SAAT1
Na/glucose
659
14
P31636
J. Biol. Chem. 268 (3), 1509-1512
cotransport
(1993)
SLC26A6
Cl/HCO exchange
738
11
NP_075062
Genomics 70 (1), 102-112
transport
(2000)
SVCT1
Na/vitamin C
598
12
NP_005838
Biochim. Biophys. Acta 1461 (1),
cotransport
1-9 (1999)
UT2
Urea (Facilitated
397
10
Q15849
FEBS Lett. 386 (2-3), 156-160
diffusion)
(1996)
Preferable transporters in the present invention are peptide transporters or organic anion transporters, and especially preferable are PepT1, Pept2, and OATP-C. The nucleotide and amino acid sequences of PepT1 and PepT2 are known (human PepT1: GenBankXM—007063, J. Biol. Chem. 270(12): 6456-6463 (1995); human PepT2: GenBank NP—066568, XM—002922, Biochem. Biophys. Acta. 1235:461-466 (1995); mouse PepT1 GenBankAF205540, Biochim. Biophys. Acta. 1492: 145-154 (2000); mouse PepT2: GenBankNM—021301, Biochim. Biophys. Res. Commun. 276: 734-741 (2000)). Furthermore, the nucleotide and amino acid sequence of OATP-C are also known (Table 1: Commun. 273(1), 251-260 (2000)). However, the transporters of the present invention are not particularly limited thereto, as long as they can be expressed on a viral envelope.
Genes encoding the transporters, for example, those listed in Table 1, are registered with the National Centre for Biotechnology Information (NCBI) under the listed accession numbers. For example, based on this sequence information, cDNA libraries or genomic libraries can be screened to obtain genes coding for transporters. More specifically, for example, cDNA or genomic libraries are screened using probes (antibodies against target transporters, or oligonucleotides that hybridise to nucleotide sequences coding for target transporters). Screening can be carried out, for example, by following the standard methods described by Sambrook et al. in Chapters 10 to 12 of “Molecular Cloning: A Laboratory Manual” (New York, Cold Spring Harbor Laboratory Press, 1989). Alternatively, genes encoding target transporters can be isolated using PCR (see e.g., Chapter 14 in the above-mentioned Sambrook et al., 1989).
As methods for expressing transporters on viral envelopes, for example, the method of WO98/46777 or Loisel et al. for expressing envelope proteins using budding baculoviruses can be used (Loisel, T. P. et al., Nature Biotech. 15: 1300-1304 (1997)). More specifically, a recombinant vector for insect cells comprising a gene encoding a transporter is constructed, and inserted, along with baculoviral DNA, into insect cells such as Sf9. The transporter encoded by the recombinant vector is then expressed on mature viral particles (virions), which are released by infected cells to the outside of cells prior to infected cell death. Thus recombinant viruses that express the transporter can be obtained.
In the present invention, a budding virus is a virus that is released from infected cells by budding. Generally, viruses covered by an envelope can bud from cells infected with these viruses, even when the cells have not been destroyed, and are released continuously. On the other hand, adenoviruses that are not covered by an envelope, and herpes viruses that are covered by a nuclear envelope, are released from the cells all at once upon their destruction. In the present invention, budding viruses are particularly preferable. In addition, hosts infected with a recombinant virus in the present invention can be suitably selected by those skilled in the art, depending on the type of virus used, so long as viral replication is possible in the host. For example, insect Sf9 cells can be used when using baculoviruses. Generally, protein expression systems using baculoviruses and insect cells may be useful because modifications such as fatty acid acetylation or glycosylation are carried out at the same time as translation or post-translation, in the same way as in mammalian cells. In addition, the expression level of heterologous proteins in such systems is greater than that in mammalian cell systems (Luckow V. A. and Summers M. D., Virol. 167: 56 (1988)).
The present invention also provides viruses that express transporters comprising transporter activity. Examples of these viruses include baculoviruses, papillomaviruses, polyomaviruses, simian virus 40 (SV40), adenoviruses, Epstein-Bar virus (EBV), and retroviruses. In the present invention, particularly preferable viruses include the AcMNPV (Invitrogen) baculovirus, and budding viruses. In addition, the transporters expressed by the viruses are preferably of a non-viral origin, for example the transporters in Table 1. Of these, peptide transporters and organic anion transporters are preferable, and Pept 1, PepT2, and OATP-C are even more preferable.
The viruses expressing transporters having transporter activity of the present invention can be obtained by, for example, culturing a host that has been infected with a recombinant virus comprising a gene that codes for a transporter. Alternatively, using methods such as the above-mentioned methods of WO98/46777 and Loisel et al (Loisel, T. P. et al., Nature Biotech. 15: 1300-1304 (1997)), a recombinant vector encoding a transporter can be inserted into an insect cell along with a baculovirus, and transporters can be expressed on the envelope of the baculovirus which is released outside of the cell. In addition, using methods like that of Strehlow et al. (D. Strehlow et al., Proc. Natl. Acad. Sci. USA. 97:4209-4214 (2000)), packaging cells such as PA317 can be infected with recombinant Moloney murine leukemia viruses, which are constructed using vectors derived from Moloney viruses introduced with transporter-encoding genes, and the transporters can be expressed on the envelope of the viruses released outside of the cells. However, the viruses of the present invention that express transporters having transporter activity are not limited to those that are constructed using the above methods. They include viruses constructed using any method as long as transporters can be expressed in viral particles or on viral surfaces.
Recombinant viruses constructed as described above can be purified using known methods. For example, known methods for purifying viruses include: augmented density gradient centrifugation (Albrechtsen et al., J. Virological Methods 28: 245-256(1990); Hewish et al., J. Virological Methods 7: 223-228 (1983)), size exclusion chromatography (Hjorth and Mereno-Lopez, J. Virological Methods 5: 151-158 (1982); Crooks et al., J. Chrom. 502: 59-68 (1990); Mento S. J. (Viagene, Inc.) 1994 Williamsburg Bioprocessing Conference), affinity chromatography using monoclonal antibodies, sulphated fucose-containing polysaccharides and the like (Najayou et al., J. Virological Methods 32: 67-77 (1991); Diaco et al., J. Gen. Virol. 67: 345-351 (1986); Fowler, J. Virological Methods 11: 59-74 (1986); TOKUSAIHYOU No. 97/032010 (Unexamined Publication of Japanese National Phase Patent Application)), and DEAE ion exchange chromatography (Haruna et al., Virology 13: 264-267 (1961)). Viruses that express transporters of the present invention are not limited to these, and can be purified using the above methods, or combinations thereof.
The present invention relates to methods for measuring the activity of transporters, which comprise expressing transporters on viral envelopes. For example, measurement of transporter activity using budding baculoviruses can be carried out by the following method. First, if necessary, a substrate to be taken into the virus by the transporters is labelled so as to be detected. For example, the substrate is labelled with radioactive substances, fluorescence, or so on. Next, the substrate is mixed with the budding baculovirus that expresses the transporter, and reacted at 37° C. After a set length of time, the reaction solution is transferred onto a filter such as a cellulose membrane. The substrate taken into the virus is separated by vacuum filtration from the substrate that was not taken up. The filter is washed several times using an ice-cold buffer, and the substrate concentration in the viruses which are trapped on the filter is determined using a liquid scintillation counter, a fluorescence detector, HPLC, or such. Nonspecific uptake can be detected by the substrate uptake into wild type viruses that do not express the transporter. In addition, nonspecific uptake can also be evaluated by carrying out experiments on substrate uptake by coexisting the substrate with transporter inhibitors, or if the substrate is radioactive, by coexisting it with an excess of unlabelled substance. Non-specific uptake can be evaluated by carrying out uptake experiments at 4° C.
As an alternative method, budding baculovirus solutions expressing a transporter can be added to a 96-well plate and incubated overnight at 4° C. to perform plate coating. Alternatively, antibodies against proteins such as gp64 protein, which is highly expressed on viral envelopes, can be added to a 96-well plate, and incubated overnight at 4° C. After this, budding baculoviruses that express the transporter are added to the plate. Antibodies against membrane proteins, such as anti-gp64 antibodies (Novagen, Clontech), can also be used to coat the plate with the viruses. A substrate is then added to the plate, and reaction begins. After a set time, the plate is washed with ice-cold buffer, and substrates that were not taken up by the viruses are removed. The amount of substrates taken up into the virus is measured using a liquid scintillation counter, fluorescence detector, HPLC, or so on. If non-specific adsorption is high, blocking can be carried out prior to measuring activity, using skim milk or such. Non-specific uptake can be detected by substrate uptake into wild-type viruses not expressing the transporter. In addition, transporter inhibitors can be coexisted with the substrate to detect non-specific uptake. Alternatively, when the substrate is a radioactive substance, non-specific uptake can also be evaluated by carrying out uptake experiments by coexisting the substrate with an excess of unlabeled substances. Furthermore, uptake experiments can be carried out at 4° C. to evaluate non-specific uptake.
Usually, cell membrane vesicles prepared from biological resources, cultured cells, and such are preserved in a deep freezer or in liquid nitrogen. However, budding baculoviruses can be preserved at 4° C., and do not require any special freezing devices. In addition, there are no complicated steps such as cell culturing, and there is no requirement for special equipment when measuring activity, as used in electrophysiological methods. Thus, budding baculovirus expression systems are simple methods for measuring transporter activity.
The methods of the present invention for measuring the transporter activity that comprise expressing transporters on viral envelopes can also be applied in searching for substances that inhibit or promote the transporter activity. In particular, methods using budding baculovirus expression systems are simple, and useful in identifying substances that inhibit or promote the transporter activity. Specifically, the methods of the present inventions produce, for example, budding baculoviruses that express target transporters. The radioactive or fluorescent substrates of those transporters are mixed with test substances, and added to the transporter-expressing viruses. Before adding the substrates, compounds can be preloaded to the viruses. Transport activity in the absence of a test substrate is taken as 100, and substances that inhibit or promote the transporter activity are searched for by using changes in activity in the presence of the test substrate as an index. Whether or not the test compound is inhibiting or promoting the transporter activity can be judged by known methods, for example, by labeling the transport target substrate (e.g. peptides in the case of peptide transporters) with a radioactive substance (such as 14C) or fluorescent substance, and then measuring the amount of that substrate that is taken up by a transporter-expressing virus, etc.
Examples of test substances in the methods of screening for substances that inhibit or promote transport activity of the transporters of the present invention include, but are not limited to, purified or crude proteins (comprising antibodies), gene library expression products, synthetic peptide libraries, cell extracts, cultured cell supernatants, products of fermentation microorganisms, marine organism extracts, vegetable extracts, synthetic low molecular weight compound libraries, peptides, non-peptide compounds, and natural compounds.
Transporters expressed on viral envelopes can be contacted with test compounds in the form of, for example, a purified protein, a form bound to a carrier, a fusion protein with another protein, or a membrane fraction. Herein, examples of carriers on which viruses can be immobilized include synthetic or natural organic high molecular weight compounds, inorganic materials such as glass beads, silica gel, alumina, and active carbon, and these materials coated with polysaccharides or synthetic high molecular weight molecules. Examples of organic high molecular weight compounds comprise a large number of compounds, including polysaccharides such as agarose, cellulose, chitin, chitosan, sepharose, and dextran, polyesters, polyvinyl cholorides, polystyrenes, polysulfones, polyether sulphones, polypropylenes, polyvinyl alcohols, polyamides, silicon resins, fluorocarbon resins, polyurethanes, polyacrylamides, and derivatives thereof. However, so long as the viruses can be immobilized, it is understood that the compositions of the compounds are not especially limited. The form of the carrier is also not particularly limited, and examples include membranes such as a plate, fibers, granules, hollow filaments, nonwoven fabrics, porous forms, and honeycomb forms. However, in the present invention, simplicity of immobilization makes commercially available plates especially preferable. By changing the form, surface area and such of these carriers, the contact area of test compounds can be controlled. Viruses can be immobilized to carriers using, for example, antibodies against the envelope proteins expressed in the viruses. In addition, immobilization onto carriers can also be achieved using streptoavidin, avidin or such when biotinylated beforehand.
The physiological function of transporters can be elucidated by searching for inhibitors or promoters of transporter activity. At the same time, those inhibitors or promoters may be applied to developing pharmaceutical agents for diseases caused by transporter abnormalities.
The present invention's budding baculoviruses that express promoters, and the envelope portions that comprise a transporter of those viruses, can be used as screening antigens or immune antigens when producing transporter antibodies. Preparation of such an antigen can be carried out, for example, according to the methods using baculoviruses described in WO98/46777.
Conventionally, in the construction of anti-transporter antibodies, it was problematic to use an active transporter as an immunogen. However, transporters that are expressed by the methods of the present invention have been confirmed to have transporter activity. Thus, an active transporter can be used as an immunogen by using the present invention's transporter-expressing viruses, or envelope portions that comprise a transporter of those viruses.
Therefore, it is extremely useful to construct antibodies using, as immunogens, the present invention's transporter-expressing viruses and envelope portions that comprise a transporter of those viruses.
Thus, the present invention provides methods for constructing anti-transporter antibodies, which methods comprise using, as immunogens, the present invention's transporter-expressing viruses or envelope portions that comprise a transporter of those viruses. The present invention also provides the antibodies constructed using these methods.
Transporter antibodies of the present invention can be constructed by those skilled in the art, using known methods where non-human animals are administered, by subcutaneous or intraperitoneal injection, several times with transporter-expressing viruses or envelope portions that comprise a transporter of those viruses.
The mammals immunized with sensitizing antigens are not particularly limited, however are preferably selected considering compatibility with parent cells used for cell fusion. Animals generally used include rodents, lagomorphs, and primates.
Examples of rodents that can be used are mice, rats, and hamsters. As lagomorphs, for example, rabbits can be used. Examples of primates are monkeys. Monkeys that can be used include catarrhines (old-world monkeys) such as cynomolgous monkeys, rhesus monkeys, hamadryas, and chimpanzees.
Animals can be immunized with a sensitizing antigen using known methods. General methods include injecting a sensitizing antigen into a mammal by subcutaneous or intraperitoneal injection. Specifically, a sensitizing antigen is diluted with an appropriate volume of Phosphate-Buffered Saline (PBS) or physiological saline, and if desired, the suspension is mixed with an appropriate volume of a conventional adjuvant, for example, Freund's complete adjuvant. After emulsification, this is applied to the mammals. In addition, after this, the sensitizing antigen that has been mixed with an appropriate volume of Freund's incomplete adjuvant is preferably applied every four to 21 days for several times. When immunizing a sensitizing antigen, an appropriate carrier can also be used. Thus immunization occurs, and the increased level of the desired antibody in the serum can be confirmed using conventional methods.
Herein, in obtaining the polyclonal antibodies against the transporters of the present invention, the increase in the level of the desired antibody in the serum is confirmed, and blood is then obtained from the mammals sensitized to the antigens. Serum can be separated from this blood using known methods. As polyclonal antibodies, serum comprising polyclonal antibodies can be used. Where necessary, fractions comprising polyclonal antibodies can be isolated from this serum, and this fraction can also be used. For example, fractions that only recognize the transporters of the present invention can be obtained using affinity columns coupled to these transporters. By purifying these fractions using a protein A or protein G column, immunoglobulin G or M can be prepared.
In obtaining monoclonal antibodies, the increased level of the desired antibody is confirmed in the mammals sensitized to the above antigen, immunocytes can be obtained from the mammals, and then subjected to cell fusion. In this case, immunocytes for cell fusion can preferably be splenocytes. As the parent cells to which the above-mentioned immunocytes are bound, mammal myeloma cells are preferable, and more preferable are myeloma cells that have acquired a characteristic for selection of fusion cells using a pharmaceutical agent.
The above-mentioned cell fusion of immunocytes and myeloma cells can be performed according to known methods, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).
Hybridomas obtained from the cell fusions can be selected by culturing in a conventional selective culture medium, for example HAT culture medium (medium comprising hypoxanthine, aminopterin, and thymidine). Culture in this HAT culture medium is carried out for a continuous period of usually several days to several weeks, a sufficient time to kill cells other than the target hybridomas (non-fusion cells). Next, conventional limiting dilution methods are carried out, and hybridomas that produce the target antibodies are screened and cloned.
In addition to obtaining the above-mentioned hybridomas by immunizing non-human animals with an antigen, human lymphocytes, for example human lymphocytes infected with EB virus, are sensitized in vitro to a virus expressing a transporter of the present invention, or to an envelope portion comprising a transporter of that virus. The sensitized lymphocytes are fused with human-derived myeloma cells that can permanently divide, for example U266. Thus, hybridomas that produce the desired human antibodies that have the activity to bind to the transporters can be obtained (Unexamined Published Japanese Patent Application No. (JP-A) Sho 63-17688).
The obtained hybridomas are transplanted into mice peritoneal cavities, and ascites are recovered from the mice. The monoclonal antibodies thus obtained can be prepared by purification using ammonium sulphate precipitation, protein A or G columns, DEAE ion exchange chromatography, affinity columns to which a transporter of the present invention has been coupled, or the like. In addition to being used for the purification and detection of the transporters of the present invention, the antibodies of the present invention can become candidates for agonists and antagonists of these transporters. Furthermore, these antibodies can also be applied to antibody therapies for diseases involving transporters of the present invention. When using the obtained antibodies for the purpose of application to the human body (antibody therapy), human antibodies and humanized antibodies are preferable due to their low antigenicity.
For example, antibody-producing cells can be obtained by immunizing transgenic animals that comprise a repertoire of human antibody genes, with a virus expressing a transporter that becomes the antigen, or a portion of the viral envelope comprising the transporter. Hybridomas produced by fusing the antibody-producing cells with myeloma cells can be used to obtain human antibodies against the transporter (see International Publication WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735, and WO96-34096).
In addition to producing antibodies by using hybridomas, immunocytes of antibody-producing sensitized lymphocytes and such that have been immortalized using oncogenes can also be used.
Monoclonal antibodies obtained in this way can also be obtained as recombinant antibodies produced using gene recombination technologies (for example, see Borrebaeck, C. A. K. and Larrick, J. W., Therapeutic Monoclonal Antibodies, UK, Macmillan Publishers Ltd., 1990). Recombinant antibodies can be produced by cloning DNA that encodes them from immunocytes such as hybridomas and antibody-producing sensitized lymphocytes, incorporating into a suitable vector, and introducing this into a host. The present invention also encompasses such recombinant antibodies.
So long as the antibodies of the present invention bind to the polypeptides of the present invention, they can also be antibody fragments, modified antibodies, etc. For example, an antibody fragment can be an Fab, F(ab′)2, Fv, or a single chain Fv (scFv) where Fvs of H chain and L chain are linked by a suitable linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A., (1998) 85, 5879-5883). Specifically, the antibody fragments can be produced by treating antibodies with an enzyme such as papain or pepsin. Alternatively, genes encoding these antibody fragments are constructed, inserted into an expression vector, and expressed in appropriate host cells (see for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).
Antibodies bound to various molecules such as polyethylene glycols (PEG), can also be used as the modified antibodies. “Antibody” in the present invention also encompasses these modified antibodies. Such a modified antibody can be obtained by chemically modifying obtained antibodies. These methods have already been established in the art.
By using known technologies, the antibodies of the present invention can be obtained as chimeric antibodies comprising non-human antibody-derived variable regions and human antibody-derived constant regions, or alternatively, as humanized antibodies comprising non-human antibody-derived complementarity determining regions (CDRs), human antibody-derived framework regions (FRs), and constant regions.
Antibodies obtained as above can be purified until homogenous. The separation and purification of antibodies used in the present invention can use conventional separation and purification methods. For example and without limitation, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography columns, filters, ultrafiltration, salt precipitation, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing and so on (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). The concentration of the above-obtained antibodies can be determined by measuring absorbance, by enzyme-linked immunosorbent assays (ELISA), etc.
Protein A columns, protein G columns, and such can be used as the columns used for affinity chromatography. For example, as the columns using protein A, Hyper D, POROS, Sepharose F.F. (Pharmacia) and so on can be used.
Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterisation: A Laboratory Course Manual. Ed Daniel R, Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographies can be carried out using liquid phase chromatography such as HPLC and FPLC.
Examples of the methods for measuring antigen-binding activities of the antibodies of the present invention include absorbance measurements, enzyme-linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), radioimmunoassay (RIA), and immunofluorescence. When using ELISA, the transporters of the present invention are added to a plate to which the antibodies of the present invention have been solid phased. Next, samples comprising a target antibody, for example the culture supernatant of antibody-producing cells or purified antibodies, are added. Secondary antibodies that recognise the antibody, which is labelled with enzymes such as alkaline phosphatase, are then added and the plate is incubated. After washing, an enzyme substrate such as p-nitrophenol phosphate is added, and antigen-binding activity can be evaluated by measuring absorbance. BIAcore (Pharmacia) can be used to evaluate the activity of the antibodies of the present invention.
Transporter-binding antibodies can be screened by ELISA using 96-well plates coated with budding baculoviruses. Antibodies against the viral antigens can be removed by ELISA using wild type viruses as the screening antigen. Alternatively, hybridoma culture supernatant and a wild type virus can be reacted, and after antibodies against the viral antigen have been removed, ELISA can be then carried out using a transporter-expressing virus as the screening antigen to acquire transporter-binding antibodies. Function-inhibiting antibodies can also be screened for from the binding antibodies. In other words, a radioactive or fluorescent substrate of the target transporter can be mixed with a solution comprising antibodies, such as hybridoma culture supernatant, and then added to a transporter-expressing virus. The solution comprising antibodies, such as hybridoma culture supernatant, can be preloaded onto the virus prior to adding the substrate. Transport activity in the absence of antibodies is taken as 100, and function-inhibiting antibodies can be screened for using, as an index, decreased activity in the presence of antibodies. Transporter topography at the cellular level can be revealed by binding antibodies to that transporter. In addition, function-inhibiting antibodies can be added to cell cultures or administered to laboratory animals to make a great contribution to the elucidation of the physiological functions of the transporters. Function-inhibiting antibodies or binding antibodies to transporters associated with disease can be applied as pharmaceutical agents.
The present invention can also be used to evaluate the ways in which transporter activity is altered by changes in amino acid sequence due to mutations, polymorphisms such as SNPs, and so on. For example, many SNPs exist in OATP-Cs, and changes in the amino acid sequence due to these SNPs have been reported (J. Biol. Chem., 276 (2001). By using the methods of the present invention to measure the transport activity of each of these OATP-Cs with altered amino acid sequences, the SNPs that influence transport activity can be identified, transporters with high activity can be screened, and so on.
In addition, after mutants have been created by artificial substitution, insertion, deletion, or addition of transporter amino acid sequences, transporter activity can be measured and transporters with high activity can be screened, or regions that influence transporter activity can be identified. Those skilled in the art can prepare transporters with substituted amino acids by using well-known methods. For example, site-specific mutagenesis and such can be used (Hashimoto-Gotoh, T. et al., Gene, 152, 271-275, (1995); Zoller, M J, and Smith, M., Methods Enzymol, 100, 468-500, (1983); Kramer, W et al., Nucleic Acids Res, 12, 9441-9456, (1984); Kramer, W and Fritz, H J., Method Enzymol, 154, 350-367, (1987); Kunkel, T A., Proc Natl Acad Sci USA, 82, 488-492, (1985); Kunkel, T A., Methods Enzymol, 85, 2763-2766, (1988)).
Further, when using the present invention, substances transported by a transporter can be used as test substances and measure transporter activity to screen for substances that are easily transported by transporters, or substances that are difficult to transport.
The present invention can also be applied to proteins other than transporters. For example, similar methods for measuring activity, screening and such can be carried out for ion channels such as sodium channels, calcium channels, potassium channels, chloride channels, cation channels, and anion channels. In this case, instead of a transporter, a channel is expressed on the viral envelope, and a substance passed through the channel can be used as a substrate. Channels that can be used in the present invention include those listed in Table 2. Thus, the present invention can be used for proteins that can transport or transmit a substance, such as transporters and ion channels (especially proteins which are expressed on membranes and can be transported or passed in a substrate-specific manner).
In addition to the above transporters and ion channels, the present invention can also be applied to G protein coupled receptors (GPCRs).
TABLE 2
Symbol
Name
Sequence ID
ACCN1
amiloride-sensitive cation channel 1,
NM_001094
neuronal (degenerin)
ACCN2
amiloride-sensitive cation channel 2,
NM_001095
neuronal
NM_020039
ACCN3
amiloride-sensitive cation channel 3, testis
NM_004769
NM_020321
NM_020322
AQP1
aquaporin 1 (channel-forming integral
NM_000385
protein, 28 kD)
ASIC4
putative acid-sensing ion channel
NM_018674
CACNA1A
calcium channel, voltage-dependent, P/Q
NM_000068
type, alpha 1A subunit
NM_023035
CACNA1B
calcium channel, voltage-dependent, L type,
NM_000718
alpha 1B subunit
CACNA1C
calcium channel, voltage-dependent, L type,
NM_000719
alpha 1C subunit
CACNA1D
calcium channel, voltage-dependent, L type,
NM_000720
alpha 1D subunit
CACNA1E
calcium channel, voltage-dependent, alpha
NM_000721
1E subunit
CACNA1F
calcium channel, voltage-dependent, alpha
NM_005183
1F subunit
CACNA1G
calcium channel, voltage-dependent, alpha
NM_018896
1G subunit
CACNA1H
calcium channel, voltage-dependent, alpha
NM_021098
1H subunit
CACNA1I
calcium channel, voltage-dependent, alpha 1I
NM_021096
subunit
CACNA1S
calcium channel, voltage-dependent, L type,
NM_000069
alpha 1S subunit
CACNA2D
calcium channel, voltage-dependent, alpha
NM_000722
2/delta subunit 1
CACNA2D
calcium channel, voltage-dependent, alpha
NM_006030
2/delta subunit 2
CACNB1
calcium channel, voltage-dependent, beta 1
NM_000723
subunit
CACNB2
calcium channel, voltage-dependent, beta 2
NM_000724
subunit
CACNB3
calcium channel, voltage-dependent, beta 3
NM_000725
subunit
CACNB4
calcium channel, voltage-dependent, beta 4
NM_000726
subunit
CACNG1
calcium channel, voltage-dependent,
NM_000727
gamma subunit 1
CACNG2
calcium channel, voltage-dependent,
NM_006078
gamma subunit 2
CACNG3
calcium channel, voltage-dependent,
NM_006539
gamma subunit 3
CACNG4
calcium channel, voltage-dependent,
NM_014405
gamma subunit 4
CACNG5
calcium channel, voltage-dependent,
NM_014404
gamma subunit 5
CACNG6
calcium channel, voltage-dependent,
NM_031897
gamma subunit 6
CACNG7
calcium channel, voltage-dependent,
NM_031896
gamma subunit 7
CACNG8
calcium channel, voltage-dependent,
AF288388
gamma subunit 8
CLCA1
chloride channel, calcium activated, family
NM_001285
member 1
CLCA2
chloride channel, calcium activated, family
NM_006536
member 2
CLCA3
chloride channel, calcium activated, family
NM_004921
member 3
CLCA4
chloride channel, calcium activated, family
NM_012128
member 4
CLCN1
chloride channel 1, skeletal muscle
NM_000083
(Thomsen disease, autosomal dominant)
CLCN2
chloride channel 2
NM_004366
CLCN3
chloride channel 3
NM_001829
CLCN4
chloride channel 4
NM_001830
CLCN5
chloride channel 5 (nephrolithiasis 2,
NM_000084
X-linked, Dent disease)
CLCN6
chloride channel 6
NM_001286
NM_021735
NM_021736
NM_021737
CLCN7
chloride channel 7
NM_001287
CLCNKA
chloride channel Ka
NM_004070
CLCNKB
chloride channel Kb
NM_000085
CLIC1
chloride intracellular channel 1
NM_001288
NM_001288
CLIC2
chloride intracellular channel 2
NM_001289
CLIC3
chloride intracellular channel 3
NM_004669
CLIC4
chloride intracellular channel 4
NM_013943
CLIC5
chloride intracellular channel 5
NM_016929
CLIC6
chloride intracellular channel 6
BG184920
CLNS1A
chloride channel, nucleotide-sensitive, 1A
NM_001293
CNGA1
cyclic nucleotide gated channel alpha 1
NM_000087
CNGA3
cyclic nucleotide gated channel alpha 3
NM_001298
CNGB1
cyclic nucleotide gated channel beta 1
NM_001297
CNGB3
cyclic nucleotide gated channel beta 3
NM_019098
DKFZP43
potassium channel modulatory factor
NM_020122
ECAC1
epithelial calcium channel 1
NM_019841
ECAC2
epithelial calcium channel 2
AJ243501
AJ243500
HCN2
hyperpolarization activated cyclic nucleotide-
NM_001194
gated potassium channel 2
HCN4
hyperpolarization activated cyclic nucleotide-
NM_005477
gated potassium channel 4
HSA24339
voltage-gated sodium channel beta-3 subunit
NM_018400
(scn3b gene)
HSA27226
calcium channel, voltage-dependent, alpha 2/
NM_018398
delta 3 subunit
KCNA1
potassium voltage-gated channel, shaker-
NM_000217
related subfamily, member 1 (episodic ataxia
with myokymia)
KCNA10
potassium voltage-gated channel, shaker-
NM_005549
related subfamily, member 10
KCNA2
potassium voltage-gated channel, shaker-
NM_004974
related subfamily, member 2
KCNA3
potassium voltage-gated channel, shaker-
NM_002232
related subfamily, member 3
KCNA4
potassium voltage-gated channel, shaker-
NM_002233
related subfamily, member 4
KCNA5
potassium voltage-gated channel, shaker-
NM_002234
related subfamily, member 5
KCNA6
potassium voltage-gated channel, shaker-
NM_002235
related subfamily, member 6
KCNA7
potassium voltage-gated channel, shaker-
NM_031886
related subfamily, member 7
KCNAB1
potassium voltage-gated channel, shaker-
NM_003471
related subfamily, beta member 1
KCNAB2
potassium voltage-gated channel, shaker-
NM_003636
related subfamily, beta member 2
KCNAB3
potassium voltage-gated channel, shaker-
NM_004732
related subfamily, beta member 3
KCNB1
potassium voltage-gated channel, Shab-
NM_004975
related subfamily, member 1
KCNB2
potassium voltage-gated channel, Shab-
NM_004770
related subfamily, member 2
KCNC1
potassium voltage-gated channel, Shaw-
NM_004976
related subfamily, member 1
KCNC3
potassium voltage-gated channel, Shaw-
NM_004977
related subfamily, member 3
KCNC4
potassium voltage-gated channel, Shaw-
NM_004978
related subfamily, member 4
KCND1
potassium voltage-gated channel, Shal-
NM_004979
related subfamily, member 1
KCND2
potassium voltage-gated channel, Shal-
NM_012281
related subfamily, member 2
KCND3
potassium voltage-gated channel, Shal-
NM_004980
related subfamily, member 3
KCNE1
potassium voltage-gated channel, Isk-related
NM_000219
family, member 1
KCNE1L
potassium voltage-gated channel, Isk-related
NM_012282
family, member 1-like
KCNE2
potassium voltage-gated channel, Isk-related
NM_005136
family, member 2
KCNE3
potassium voltage-gated channel, Isk-related
NM_005472
family, member 3
KCNF1
potassium voltage-gated channel, subfamily
NM_002236
F, member 1
KCNG1
potassium voltage-gated channel, subfamily
NM_002237
G, member 1
KCNG2
potassium voltage-gated channel, subfamily
NM_012283
G, member 2
KCNH1
potassium voltage-gated channel, subfamily
NM_002238
H (eag-related), member 1
KCNH2
potassium voltage-gated channel, subfamily
NM_000238
H (eag-related), member 2
KCNH3
potassium voltage-gated channel, subfamily
AB033108
H (eag-related), member 3
KCNH4
potassium voltage-gated channel, subfamily
NM_012285
H (eag-related), member 4
KCNH5
potassium voltage-gated channel, subfamily
U69185
H (eag-related), member 5
KCNIP1
Kv channel-interacting protein 1
NM_014592
KCNIP2
Kv channel-interacting protein 2
NM_014591
KCNJ1
potassium inwardly-rectifying channel,
NM_000220
subfamily J, member 1
KCNJ10
potassium inwardly-rectifying channel,
NM_002241
subfamily J, member 10
KCNJ11
potassium inwardly-rectifying channel,
NM_000525
subfamily J, member 11
KCNJ12
potassium inwardly-rectifying channel,
NM_021012
subfamily J, member 12
KCNJ13
potassium inwardly-rectifying channel,
AJ007557
subfamily J, member 13
KCNJ14
potassium inwardly-rectifying channel,
NM_013348
subfamily J, member 14
KCNJ15
potassium inwardly-rectifying channel,
NM_002243
subfamily J, member 15
KCNJ16
potassium inwardly-rectifying channel,
NM_018658
subfamily J, member 16
KCNJ2
potassium inwardly-rectifying channel,
NM_000891
subfamily J, member 2
KCNJ3
potassium inwardly-rectifying channel,
NM_002239
subfamily J, member 3
KCNJ4
potassium inwardly-rectifying channel,
NM_004981
subfamily J, member 4
KCNJ5
potassium inwardly-rectifying channel,
NM_000890
subfamily J, member 5
KCNJ6
potassium inwardly-rectifying channel,
NM_002240
subfamily J, member 6
KCNJ8
potassium inwardly-rectifying channel,
NM_004982
subfamily J, member 8
KCNJ9
potassium inwardly-rectifying channel,
NM_004983
subfamily J, member 9
KCNJN1
potassium inwardly-rectifying channel,
NM_002244
subfamily J, inhibitor 1
KCNK1
potassium channel, subfamily K, member 1
NM_002245
(TWIK-1)
KCNK10
potassium channel, subfamily K, member 10
NM_021161
KCNK12
potassium channel, subfamily K, member 12
NM_022055
KCNK13
potassium channel, subfamily K, member 13
NM_022054
KCNK2
potassium channel, subfamily K, member 2
AF004711
(TREK-1)
KCNK3
potassium channel, subfamily K, member 3
NM_002246
(TASK-1)
KCNK4
potassium inwardly-rectifying channel, sub-
NM_016611
family K, member 4
KCNK5
potassium channel, subfamily K, member 5
NM_003740
(TASK-2)
KCNK6
potassium channel, subfamily K, member 6
NM_004823
(TWIK-2)
KCNK7
potassium channel, subfamily K, member 7
NM_005714
KCNK9
potassium channel, subfamily K, member 9
NM_016601
(TASK-3)
KCNMA1
potassium large conductance calcium-acti-
NM_002247
vated channel, subfamily M, alpha member 1
KCNMB1
potassium large conductance calcium-acti-
NM_004137
vated channel, subfamily M, beta member 1
KCNMB2
potassium large conductance calcium-acti-
NM_005832
vated channel, subfamily M, beta member 2
KCNMB3
potassium large conductance calcium-acti-
NM_014407
vated channel, subfamily M beta member 3
KCNMB3L
potassium large conductance calcium-acti-
NM_014406
vated channel, subfamily M, beta member
3-like
KCNMB4
potassium large conductance calcium-acti-
NM_014505
vated channel, subfamily M, beta member 4
KCNN1
potassium intermediate/small conductance
NM_002248
calcium-activated channel, subfamily N,
member 1
KCNN2
potassium intermediate/small conductance
NM_021614
calcium-activated channel, subfamily N,
member 2
KCNN3
potassium intermediate/small conductance
NM_002249
calcium-activated channel, subfamily N,
member 3
KCNN4
potassium intermediate/small conductance
NM_002250
calcium-activated channel, subfamily N,
member 4
KCNQ1
potassium voltage-gated channel, KQT-like
NM_000218
subfamily, member
KCNQ2
potassium voltage-gated channel, KQT-like
NM_004518
subfamily, member
KCNQ3
potassium voltage-gated channel, KQT-like
NM_004519
subfamily, member
KCNQ4
potassium voltage-gated channel, KQT-like
NM_004700
subfamily, member
KCNQ5
potassium voltage-gated channel, KQT-like
NM_019842
subfamily, member
KCNS1
potassium voltage-gated channel, delayed-
NM_002251
rectifier, subfamily S, member 1
KCNS2
potassium voltage-gated channel, delayed-
AB032970
rectifier, subfamily S, member 2
KCNS3
potassium voltage-gated channel, delayed-
NM_002252
rectifier, subfamily S, member 3
KIAA0439
homolog of yeast ubiquitin-protein ligase
AB007899
Rsp5; potential epithelial sodium channel
regulator
KIAA1169
two-pore channel 1, homolog
NM_017901
KV8.1
neuronal potassium channel alpha subunit
NM_014379
LOC64181
two pore potassium channel KT3.3
NM_022358
OTRPC4
vanilloid receptor-related osmotically
NM_021625
activated channel; OTRPC4 protein
P2RX1
purinergic receptor P2X, ligand-gated ion
NM_002558
channel, 1
P2RX2
purinergic receptor P2X, ligand-gated ion
NM_012226
channel, 2
NM_016318
P2RX3
purinergic receptor P2X, ligand-gated ion
NM_002559
channel, 3
P2RX4
purinergic receptor P2X, ligand-gated ion
NM_002560
channel, 4
P2RX5
purinergic receptor P2X, ligand-gated ion
NM_002561
channel, 5
P2RX7
purinergic receptor P2X, ligand-gated ion
NM_002562
channel, 7
SCN10A
sodium channel, voltage-gated, type X, alpha
NM_006514
polypeptide
SCN11A
sodium channel, voltage-gated, type XI,
AF188679
alpha polypeptide
SCN12A
sodium channel, voltage-gated, type XII,
NM_014139
alpha polypeptide
SCN1A
sodium channel, voltage-gated, type I, alpha
AF225985
polypeptide
SCN1B
sodium channel, voltage-gated, type I, beta
NM_001037
polypeptide
SCN2A2
sodium channel, voltage-gated, type II, alpha
NM_021007
2 polypeptide
SCN2B
sodium channel, voltage-gated, type II, beta
NM_004588
polypeptide
SCN3A
sodium channel, voltage-gated, type III,
AF225987
alpha polypeptide
SCN4A
sodium channel, voltage-gated, type IV,
NM_000334
alpha polypeptide
SCN5A
sodium channel, voltage-gated, type V, alpha
NM_000335
polypeptide (long (electrocardiographic) QT
syndrome 3)
SCN6A
sodium channel, voltage-gated, type VI,
NM_002976
alpha polypeptide
SCN8A
sodium channel, voltage gated, type VIII,
NM_014191
alpha polypeptide
SCN9A
sodium channel, voltage-gated, type IX,
NM_002977
alpha polypeptide
SCNN1A
sodium channel, nonvoltage-gated 1 alpha
NM_001038
SCNN1B
sodium channel, nonvoltage-gated 1, beta
NM_000336
(Liddle syndrome)
SCNN1D
sodium channel, nonvoltage-gated 1, delta
NM_002978
SCNN1G
sodium channel, nonvoltage-gated 1, gamma
NM_001039
TALK-1
pancreatic 2P domain potassium channel
NM_032115
TALK-1
TASK-4
potassium channel TASK-4; potassium
NM_031460
channel TALK-2
TRPC1
transient receptor potential channel 1
NM_003304
TRPC2
transient receptor potential channel 2
X89067
TRPC3
transient receptor potential channel 3
NM_003305
TRPC4
transient receptor potential channel 4
NM_016179
TRPC5
transient receptor potential channel 5
NM_012471
TRPC6
transient receptor potential channel 6
NM_004621
TRPC7
transient receptor potential channel 7
NM_003307
VDAC1
voltage-dependent anion channel 1
NM_003374
VDAC1P
voltage-dependent anion channel 1
AJ002428
pseudogene
VDAC2
voltage-dependent anion channel 2
NM_003375
VDAC3
voltage-dependent anion channel 3
NM_005662
trp7
putative capacitative calcium channel
NM_020389
Herein below, the present invention will be specifically described using Examples, however, it is not to be construed as being limited to thereto.
1. Preparation of PepT1-expressing Budding Baculoviruses
A full-length PepT1 gene was isolated from a human kidney library using PCR. By inserting the full-length human PepT1 gene into pBlueBacHis2A (Invitrogen), the pBlueBacHis-PepT1 transfer vector was constructed. A Bac-N-Blue transfection kit (Invitrogen) was then used to introduce this transfer vector into Sf9 cells, along with Bac-N-Blue DNA. Thus, a recombinant virus for the expression of human PepT1 was constructed. Specifically, 4 μg of pBlueBacHis-PepT1 was added to Bac-N-Blue DNA, and then 1 mL of Grace's medium (GIBCO) and 20 μL of cell FECTIN reagent was added. This was mixed, incubated for 15 minutes at room temperature, and then added drop-by-drop to 2×106Sf9 cells washed once with the Grace's medium. After incubating for four hours at room temperature, 2 mL of complete medium (Grace's medium which comprises 10% fetal bovine serum (Sigma), 100 units/mL penicillin, and 100 μg/mL streptomycin (GIBCO-BRL)) was added and cultured at 27° C. Recombinant viruses for expressing human PepT1, which were constructed by homologous recombination, were cloned twice according to the instructions attached to the kit. A virus stock of the recombinant viruses was thus obtained.
Construction of budding-type viruses that express human PepT1 was carried out as follows. Specifically, 500 mL of Sf9 cells (2×106/mL) were infected with the recombinant viruses prepared as above, so as to achieve MOI=5. After culturing at 27° C. for three days, the culture supernatant was centrifuged for 15 minutes at 800×g, and the cells and cell debris were removed. The supernatant recovered by centrifugation was centrifuged at 45,000×g for 30 minutes, and the precipitate was then suspended in PBS. The cellular components were removed by centrifuging for another 15 minutes at 800×g. The supernatant was again centrifuged at 45,000×g for 30 minutes, and the precipitate was again suspended in PBS. This suspension was the budding virus fraction. Expression of PepT1 in the virus and on the Sf-9 cell membrane was confirmed by Western analysis using anti-His antibodies. In addition, protein concentration was measured using Dc Protein Assay kit (Bio-Rad), with BSA as the standard.
2. PepT1 Functional Analysis
14C glycylsarcosine was diluted with HBSS (pH6.0) to a final concentration of 50 μM, and used as a substrate solution. 40 μL of viral solution (100 μg protein) was preincubated at 37° C. for 30 minutes. 160 μL of substrate solution that had been preheated to 37° C. was added, and the reaction was started. After one minute, 1 mL of ice-cold HBSS (pH 7.4)(hereinbelow also called “quenching solution”) was added, and the reaction was stopped. The virus-comprising reaction solution was immediately vacuum filtered using a mixed cellulose membrane filter, and washed twice with 5 mL of the quenching solution. The membrane filter was transferred to a liquid scintillation vial, 5 mL of clear-zol I was added, and the filter was dissolved. After the dissolving, a liquid scintillation counter was used to measure radioactivity on the filter. Non-specific adsorption to the filter was measured in the same way for systems where the quenching solution was added before adding the substrate solution to the viral solution, and values thus obtained were subtracted from the counts for each experiment.
The PepT1 activity of the PepT1-expressing virus with a His-tag added at its N-terminal is shown in
1. Preparation of PepT2-expressing Budding Baculoviruses
The full-length PepT2 gene was isolated from a human kidney library. PCR was used to integrate the gene encoding the full-length human PepT2 into pBlueBacHis2A (Invitrogen), and a full-length PepT2 transfer vector (pBlueBac) was constructed. This vector was introduced into Sf-9 cells along with the viral DNA. After cloning the recombinant virus constructed by homologous recombination, a stock with a high recombinant virus activity was constructed. Sf-9 cells were infected with the stock virus, and after culturing for a certain period, PepT2 was expressed in the virus and on the membrane of Sf-P cells. PepT2 expression in the virus and on the membrane of the Sf-9 cells was confirmed by Western analysis using anti-His antibodies. More specifically, except for using the PepT2 gene, operations were carried out according to the methods described in Example 1.
2. PepT2 Functional Analysis
3H glycylsarcosine was diluted with HBSS (pH6.0) to a final concentration of 0.8 μM, and used as a substrate solution. 40 μL of viral solution (100 μg protein) was preincubated at 37° C. for 30 minutes. 160 μL of the substrate solution preheated to 37° C. was added to commence the reaction. After one minute, 1 mL of the quenching solution was added, and the reaction was stopped. The virus-comprising reaction solution was immediately vacuum filtered using a mixed cellulose membrane filter, and washed twice with 5 mL of the quenching solution. The membrane filter was transferred to a liquid scintillation vial, 5 mL of clear-sol I was added, and the filter was dissolved. After the dissolving, a liquid scintillation counter was used to measure radioactivity on the filter. The quenching solution was added before adding the substrate solution to the viral solution, and similar manipulations were performed. Non-specific adsorption to the filter was measured and the obtained value was subtracted from the counts for each experiment.
The PepT2 activity of the PepT2-expressing virus with a His-tag added to its N-terminal is shown in
1. Preparation of OATP-C Expressing Baculoviruses
cDNA encoding wild type human OATP-C (OATP-C WT) was cloned as follows. Specifically, adult human liver-derived cDNA was used as a template, and the OATP-C WT cDNA was divided into two fragments and amplified using PCR with the following primer combinations:
5′ side
OAHC17 primer.
5′ gat ggt acc aaa
(SEQ ID NO: 1)
ctg agc atc aac aac
aaa aac 3′
OAHC18 primer:
5′ gat ggt acc cat
(SEQ ID NO: 2)
cga gaa tca gta gga
gtt atc 3′
3′ side
OAHC21 primer:
5′ gat ggt acc tac
(SEQ ID NO: 3)
cct ggg atc tct gtt
ttc taa 3′
OAHC22 primer:
5′ gat ggt acc gtt
(SEQ ID NO: 4)
tgg aaa cac aga agc
aga agt 3′
Each of these fragments were subcloned to pT7Blue-T vector (Novagen), and clones without PCR errors were selected. Both were linked at the BglII site which exists in an overlapping region, and then cleaved at the KpnI site that exists on both ends. After incorporation at the KpnI site of pcDNA3 vector (Invitrogen), pcDNA3/OATP-C WT was obtained.
Next, with pcDNA3/OATP-C WT as a template, in vitro mutageneis using GeneEditor™ (Promega) was used to prepare cDNAs coding for OATP-C N130D in which the 130th asparagine was mutated to aspartic acid, and OATP-C V174A in which the 174th valine was mutated to alanine. The primers used for mutagenesis were as follows:
Primer for OATP-C N130D:
5′ gaa act aat atc gat tca tca gaa
(SEQ ID NO: 5)
aat 3′
Primer for OATP-C V174A:
5′ atg tgg ata tat gcg ttc atg ggt
(SEQ ID NO: 6)
aat 3′
The primers for use in mutagenesis and the selection primers included in kits (for bottom strand use) were both annealed to the template plasmid DNA, which had been made into a single strand. Thus, a new DNA strand was constructed. This was introduced into E. coli, and GeneEditor™ antibiotic-resistant clones were obtained. These clones were sequenced and clones containing mutations were thus selected (pcDNA3/OATP-C N130D and pcDNA3/OATP-C V174A).
Next, using pcDNA3/OATP-C WT, pcDNA3/OATP-C N130D and pcDNA3/OATP-CV174A as respective templates, PCR was carried out using the primers below, thus amplifying the respective cDNAs with SalI sites on each end.
C45 primer:
5′ gat gtc gac tta aca
(SEQ ID NO 7)
atg tgt ttc act 3′
C58 primer:
5′ gat gtc gac tat gga
(SEQ ID NO: 8)
cca aaa tca aca t 3′
These were digested with SalI, and then inserted into the SalI site of the pBlueBac His2A vector (Invitrogen). Thus transfer vectors encoding each OATP-C protein with a His-tag attached at the N-terminal were constructed (pBlueBac His2A/OATP-C WT, pBlueBac His2A/OATP-C N130D, pBlueBac His2A/OATP-C V174A).
Using Bac-N-Blue transfection kit (Invitrogen), these vectors were introduced into Sf-9 cells along with viral DNA. After five to eight days, plaque assays were used to clone the recombinant viruses in the culture supernatant. The viruses were then amplified, and a stock of highly active recombinant viruses was prepared. Sf-9 cells were infected with the stock viruses at MOI=1. After four days, recombinant viruses were recovered from the culture supernatant. OATP-C expression on the viral envelope was confirmed by Western analysis using anti-His antibodies.
2. OATP-C Functional Analysis.
3H estrone sulphate conjugate was diluted with HBSS (pH7.4) to a final concentration of 10 nM, and used as a substrate solution. 20 μL of viral solution (50 μg protein) was preincubated at 37° C. for 30 minutes. 180 μL of the substrate solution preheated to 37° C. was added, and the reaction was started. After one minute, 1 mL of ice-cold HBSS (pH7.4) (hereinafter referred to as “quenching solution”) was added, and the reaction was stopped. The virus-comprising reaction solution was immediately vacuum filtered using a mixed cellulose membrane filter, and washed twice with 5 mL of the quenching solution. The membrane filter was transferred to a liquid scintillation vial, 5 mL of clear-sol I was added, and the filter was dissolved. After the dissolving, a liquid scintillation counter was used to measure radioactivity on the filter. To measure non-specific adsorption to the filter, the reaction quenching solution was added before adding the substrate solution and similar manipulations were performed. The obtained value was subtracted from the counts for each experiment.
The activity of 3H estrone sulphate conjugate uptake is shown in
14C glycylsarcosine was diluted with HBSS (pH 6.0) to a final concentration of 50 μM, and used as a substrate solution. In addition, a mouse monoclonal antibody recognising the extracellular region of human PepT1 was diluted with PBS to a final concentration of 200 μg/mL, and used as an antibody solution. 20 μL (50 μg protein) of solution of budding baculoviruses expressing PepT1 with a His-tag added at the N-terminal was mixed with 20 μL of the antibody solution and incubated for one hour at 37° C. 160 μL of substrate solution preheated to 37° C. was added, and the reaction was started. After one minute, 1 mL of ice-cold HBSS (pH 7.4) (below also called “quenching solution”) was added, and the reaction was stopped. The virus-comprising reaction solution was immediately vacuum filtered using a mixed cellulose membrane filter, and washed twice with 5 mL of the quenching solution. The membrane filter was transferred to a liquid scintillation vial, 5 mL of clear-sol I was added, and the filter was dissolved. After the dissolving, a liquid scintillation counter was used to measure radioactivity on the filter. Non-specific adsorption to the filter was measured by adding the reaction quenching solution before adding the substrate solution to the viral solution, and performing similar manipulations. The obtained value was subtracted from the counts for each experiment.
The PepT1 activity inhibition by the anti-human PepT1 monoclonal antibodies is shown in
The present invention provides viruses that express transporters having transporter activity, and by using these viruses, transporter activity can be measured with a high sensitivity and less background level than in the past. Thus, it is expected that by employing the methods of the present invention, identification of transport substrates and driving force of transporters, and functional analysis such as kinetic analysis can be carried out more easily and accurately than before. In addition, by using such viruses, substances that inhibit or promote the transport activities of transporters expressed on the viral envelopes can be screened. Since transporters have also been reported to be involved in the transport of drugs into cells, substances that inhibit or promote the activities of transporters associated with diseases can become candidates for new pharmaceutical agents. Furthermore, by using the methods of the present invention for analysis of SNPs in transporter-encoding genes, functional changes due to transporter SNPs can be measured over a more extensive range of substrates. Application to tailor-made therapies is also possible since response to a drug can be analyzed for each individual.
Tsuchiya, Masayuki, Ohtomo, Toshihiko, Saitoh, Ryoichi
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